Podocyte foot processes and the interposed slit diaphragms form the final barrier to protein loss, explaining why podocyte injury is typically associated with marked proteinuria. The highly dynamic foot process actin cytoskeleton is linked to the slit diaphragm and proteins regulating podocyte actin dynamics are therefore of critical importance for structural maintenance and sustained function of the glomerular filter. Synaptopodin was first described by the P.I. as the founding member of a novel class of actin associated proteins highly expressed in podocytes and telencephalic dendrites Based on the data obtained with this grant, we established that synaptopodin stabilizes the kidney filter by blocking the re-organization of the podocyte actin cytoskeleton into a migratory phenotype We also established that the calcineurin inhibitor cyclosporine A protects against proteinuria by stabilizing synaptopodin steady-state levels in podocytes. Here we propose to test our central hypothesis that the activation of the tyrosine kinase Src in podocytes contributes to the pathogenesis of proteinuria by promoting the degradation of synaptopodin, thereby shifting the balance of Rho GTPase activity towards pro-migratory Rac1 signaling. To test this hypothesis we propose the following three Specific Aims. The first Aim will define the molecular mechanism by which Src induced degradation of synaptopodin affects the actin cytoskeleton in podocytes. Specific Aim two seeks to explore the regulation of Rac1 activation in podocytes by synaptopodin. The third Aim will establish the contribution of synaptopodin degradation to the pathogenesis of podocyte foot process effacement proteinuria in vivo. If our hypothesis is correct, the work proposed here will have broad significance because it will provide us with a better understanding of the biological mechanism underlying the dynamic re-organization of the podocyte actin cytoskeleton in normal and proteinuric kidneys. This should in the long-term enable us to develop novel, selective podocyte- protective therapies that tackle proteinuria by promoting the synaptopodin-dependent preservation of the normal foot process architecture. PUBLIC HEALTH RELEVANCE: Our work, funded by this grant, has revealed a novel synaptopodin-dependent signaling pathway for the regulation of the podocyte function in health and disease. This detailed understanding of a synaptopodin-specific anti-proteinuric effect validates the podocyte as target of choice for the treatment of proteinuria. The goal of this application is to further define the molecular mechanisms whereby the stabilization of synaptopodin protein expression protects against proteinuria, thereby paving the road for the development of novel anti-proteinuric treatment options for which there is a critical need in the clinic. !